Catalytic radiofluorination

ABSTRACT

One aspect of the present invention relates to a method of preparing radiofluorinated substituted alkyl, cycloalkyl, aryl, and alkenyl compounds. In a preferred embodiment, potassium fluoride-18 is used. Another aspect of the invention relates to arylammonium compounds containing fluorine-18 that are useful as imaging agents. In certain embodiments, the ammonium compound is a tetraaryl ammonium salt. Another aspect of the invention relates to arylsulfonium compounds containing fluorine-18 that are useful as imaging agents. In certain embodiments, the sulfonium compound is a triaryl sulfonium salt. Another aspect of the present invention relates to a method of obtaining a positron emission image of a mammal, comprising the steps of administering to a mammal a compound of the invention, and acquiring a positron emission spectrum of the mammal.

RELATED APPLICATIONS

This application is a divisional application and claims the benefit ofpriority to U.S. patent application Ser. No. 13/602,810, filed Sep. 4,2012, now U.S. Pat. No. 8,900,550, which is a continuation applicationand claims the benefit of priority to U.S. patent application Ser. No.11/506,603, filed Aug. 18, 2006, now U.S. Pat. No. 8,257,680, which is acontinuation-in-part of and claims the benefit of priority to U.S.patent application Ser. No. 11/065,345, filed Feb. 24, 2005, now U.S.Pat. No. 7,632,485, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/547,206, filed Feb. 24, 2004; the contents ofall of which are incorporated by reference.

BACKGROUND OF THE INVENTION

Contemporary medical imaging depends largely upon the use ofradioisotopes. One of the first clinically-employed radioisotopes wastechnetium (Tc). This element was first administered to a human subjectin 1961 in the form of Na^(99m)TcO₄. Other radioisotopes, includinghalogens, such as ¹²⁵I, ¹³¹I and ⁸²Br, and isotopes of various metalradionuclides of lead, gallium, rhenium, arsenic and copper, have alsobeen explored as potential imaging agents. Medical imaging is used in avariety of medical applications, including imaging of the brain, tumors,and components of the cardiovascular system.

Blood flow imaging agents are currently the most important tool fordetermining heart function. Tl-201, Tc-99-MIBI and Tc-99-tetrofosmin arein routine use for myocardial imaging at rest and after exercise. Theseagents are very useful but are not optimal. These tracers are SinglePhoton Imaging agents and their resolution is limited to the propertiesof SPECT imaging cameras and technology. However, fluorine-18 can bedetected by Positron Emission Tomography imaging technology which hasseveral advantages including higher resolution and corrections for theemitted radiation attenuation. In fact, the number of PET cameras andimaging centers are growing rapidly in response to the superiorperformance properties of fluorine-18.

F-18 is one of the most useful positron emitting radionuclides currentlybeing used in clinical nuclear medicine diagnosis. For example, 2-[F-18]FDG (2-[F-18]-fluoro-2-deoxy-D-glucose) is the radiopharmaceutical ofchoice for the diagnosis of several cancers and brain disorders. Thisradiopharmaceutical agent produces superior high-resolution images andquantitative regional uptake of tissues. The 110-min half-life offluorine-18 allows production and distribution of 2-[F-18] FDG tonuclear medicine facilities near a cyclotron center. The relatively longphysical half-life of fluorine-18 also permits PET studies of moderatelyslow physiological process. Decay of fluorine-18 is largely by positronemission (97%), and the emitted positron is of relatively low energy(maximum 0.635 MeV) and thus has a short mean range (2.39 mn in water).Fluorine-18 is readily available from both particle accelerators andnuclear reactors using a wide variety of nuclear reactions, and can beproduced at specific activities approaching the theoretical limit of1.171×10⁹ Ci/mmol.

In addition to their superior medical imaging properties, fluorine atomsare a component of many pharmaceutical compounds. Fluorine can functionas a substitute for a hydrogen atom in many biologically activemolecules without substantially altering their properties, as done inthe case of 2-deoxy-D-glucose.

Despite the utility of F-18, there are only a very small number ofmethods to introduce F-18 into organic molecules. To date, theintroduction of F-18 to a single bond was made via an exchange reactionon mesylate or triflate. Alternatively, F-18 could be introduced onto aC—C double bond or aromatic ring via an appropriate tin compound and[F-18]F₂ or using anhydrous [F-18]fluoride on an electron withdrawingactivated ring. The exchange reaction is carried out by treating themesylate or triflate with a mixture of F-18, potassium carbonate, andcrown ether, such as Kryptofix. 2-[F-18] FDG is the best example of thatreaction. Other reactions using, [F-18]-F₂, [F-18]XeF₂, [F-18]DAST,[F-18]triethylammonium fluoride were also reported for specificradiolabeling. Radiofluorination of tributyltin-substituted double bondsand aromatic rings used [F-18]F₂ as a reagent. However, the specificactivity of these radiofluorinations is very low due to the cold F₂carrier. Radiofluorination of a nitro moiety on an activated aromaticring with F-18 anhydrous fluoride was also reported. However, mostfluorine-containing drugs are not activated with electron withdrawinggroups, such as nitro, aldehyde, ketone, ester or others; therefore,this reaction is not applicable for a large number of compounds.

There is an urgent need for the development of new agents that canimprove the diagnosis of heart disease by understanding the molecularbehavior, physiology, anatomy, and function of the myocardium. However,many biologically-active molecules, drugs, receptor ligands, peptides,and proteins are not readily available for clinical nuclear medicine dueto the limitations inherent in the methods used to install F-18.Therefore, the need exists for a new method for labeling a compound withF-18 which is amenable to a wide variety of organic substrates.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of preparingfluorosubstituted alkyl, cycloalkyl, aryl, and alkenyl compounds. Incertain embodiments, anhydrous potassium fluoride is reacted with analkyl or cycloalkyl sulfonate, e.g., a mesylate. In certain embodiments,anhydrous potassium fluoride is reacted with a nitroaromatic compound.In a preferred embodiment, the reaction is conducted in the presence ofKryptofix. In a preferred embodiment, potassium fluoride-18 is used. Inanother embodiment, the reaction is conducted under microwave radiationfor a substantially shorter reaction time either as a neat mixture, withsolvent, or with or without catalyst.

In one embodiment, a nitro group is exchanged with, for example, F-18.Separating the compounds on HPLC is difficult because Rf and elutiontimes are similar. Therefore, separation and purification is complexresulting in lower radiolabeling yield and specific activity. Thepresent invention includes, in part, an alternative route which quicklyreduces (˜2 minutes) the remaining nitro compound precursors in thereaction mixture. The reduction gives high yields of the amine. Aminesare very different in their properties from the fluorine compounds andcan be readily separated.

Another aspect of the invention relates to piperazine compoundscontaining fluorine-18 that are useful as imaging agents. In certainembodiments, the piperazine compounds contain a quaternary amine. Incertain embodiments, the piperazine compounds are N-substituted by acycloalkyl or aryl group. In a preferred embodiment, the piperazinecompounds are substituted at the 4-position with a phenyl group andsubstituted at the I-position by a fluorocycloalkyl group.

Another aspect of the invention relates to arylphosphonium compoundscontaining fluorine-18 that are useful as imaging agents. In certainembodiments, the phosphonium compound is a tetraaryl phosphonium salt.In a preferred embodiment, the arylphosphonium compound is atetraphenylphosphonium salt.

Another aspect of the invention relates to arylammonium compoundscontaining fluorine-18 that are useful as imaging agents. In certainembodiments, the ammonium compound is a tetraaryl ammonium salt. In apreferred embodiment, the arylammonium compound is a tetraphenylammoniumsalt.

Another aspect of the invention relates to arylsulfonium compoundscontaining fluorine-18 that are useful as imaging agents. In certainembodiments, the sulfonium compound is a triaryl sulfonium salt. In apreferred embodiment, the arylsulfonium compound is a triphenylsulfoniumsalt.

Another aspect of the present invention relates to a method of obtaininga positron emission image of a mammal, comprising the steps ofadministering to a mammal a compound of the invention, and acquiring apositron emission spectrum of the mammal. In a preferred embodiment, thecompound of the invention is a piperazine substituted with fluorine-18.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts various metal-activated arenes.

FIG. 2 depicts biodistribution data in rats for[F-18]1-(4-Fluorocyclohexyl)-1-Methyl-4-Phenylpiperazium (Example 14).

FIG. 3 depicts cell distribution data of [I-125]TPPI (Example 14).

FIG. 4 depicts biodistribution of [I-125]TPPI in rats implanted withC6-BAG glioma (Example 14).

FIG. 5 depicts biodistribution in rats for[F-18](4-fluorophenyl)triphenylphosphonium (TPPF).

FIG. 6 depicts images of rat-heart slices (coronal (left), transverse(center), suggital (right)) obtained with a MicroPET camera afterinjection of [F-18](4-fluorophenyl)triphenyl-phosphonium salt{[F-18]FTPP}.

FIG. 7 depicts a time activity curve of rat heart obtained after IVinjection of [F-18](4-fluorophenyl)triphenyl-phosphonium nitrate (normalstate yellow) and after second injection of[F-18](4-fluorophenyl)triphenyl-phosphonium nitrate and an adenosinebolus injection (blue). A 29% increase in flow was observed.

FIG. 8 depicts time-dependent activity of FTTP in blood pool inside theleft ventricle (bottom) and in heart muscle (top).

FIG. 9 depicts time-dependent activity of FTTP in blood pool inside theleft ventricle and in heart muscle.

FIG. 10 depicts N-13-ammonia images and time activity curves before andafter LAD occlusion.

FIG. 11 depicts FTTP tomogramps and time activity curves of a rabbitbefore and after LAD occlusion.

FIG. 12 depicts N-13-ammonia and FTTP images and their correspondingtime activity curves post LAD occlusion (ROI analysis).

DETAILED DESCRIPTION OF THE INVENTION

Radioisotope Imaging

Thallium-201, Tc-99-sestamibi and tetrofosmin are currently the mostwidely used radiopharmaceutical for clinical evaluation of myocardialperfusion. However, the widespread use of PET (Position EmissionTomography) technology and the limitations of these agents with respectto their properties as SPECT agents has stimulated the search for moresuitable PET tracers. Most approaches for developing a myocardialperfusion agent have generally involved the incorporation of iodine-123or technetium-99m into a cationic moiety, thereby taking advantage ofthe better radionuclidic properties of iodine and technetium whilepotentially retaining the distributional properties of monocationicthallium(I) species. Numerous publications have described the abovesingle-photon-emission computed tomographic (SPECT) commercial agentsfor research and clinical diagnosis of heart disease. An alternativemethod for designing a blood flow agent is the use of a freelydiffusible agent like antipyrine. However, this approach is oftenavoided due the difficulties in modeling and the required fast kineticdata collection for these agents as compared to agents that are freelydiffusible and significantly trapped in the tissue.

The widespread use of F-18 FDG and the exponential increase in PETscanners focused our effort on developing an appropriate F-18 lipophiliccationic agent. We developed two series of agents based on themodification of charged piperazinium salts and of tetraphenylphosphoniumsalts. The synthesis and F-18 radiolabeling of these salts is nottrivial due to the need for a suitable precursor and the appropriateconditions for radiolabeling. Here we describe several specificstructures and their analogs as blood flow agents. We have devisedsyntheses and radiolabeling procedures for these agents.

Catalytic Radiohalogenation

The catalytic radiohalogenation reaction of the invention involvesreacting anhydrous potassium halide, a crown ether, and an organiccompound that has a leaving group. In a preferred embodiment, theradiohalogenation is a radiofluorination. The reaction proceeds bysubstitution of the leaving group by the halide. Importantly, thereaction of the present invention does not require that the compoundcontain an activating group to enhance the reactivity of the leavinggroup. For example, the radiofluorination reaction of the inventionworks on unactivated nitroaryl groups, e.g., nitrophenyl groups. Thefact that the radiofluorination reaction of the invention works on both“activated” and “unactivated” compounds is an important breakthrough andwill allow for the facile preparation of many ¹⁸F-labeled compoundsuseful for medical imaging.

In addition, the reaction can be conducted in the presence or absence ofsolvent. For reactions conducted in the presence of the solvent, thereaction should be amenable to most organic solvents which do not have ahydroxyl group which might react with the substrates of the reaction. Arepresentative selection of suitable solvents includes acetonitrile,dimethylacetamide, dimethylformamide, dimethylsulfoxide, dioxane,benzene, toluene, xylene, ethylbenzene diglyme, dimethoxyethane (glyme),diethylene glycol dibutyl ether, triethylene glycol dimethyl ether,tetraethylene glycol dimethyl ether, polyethylene glycol dimethylethers, diethylene glycol dibutyl ether, polyethylene glycol dibutylethers, heptane, octane, butylacetate and the like. The ideal solventfor a particular reaction can be determined by one of ordinary skill inthe art taking into consideration the preferred temperature of thereaction, the boiling point of the solvent, and the solubilities of thesubstrates in the solvent.

In certain embodiments, the crown either is Kryptofix. A variety ofKryptofix compositions will work in the instant invention including:1,4,10-Trioxa-7,13-diaza-cyclopentadecane (Kryptofix® 21),4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix®222), 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo[8.8.5]tricosane(Kryptofix® 221), and4,7,13,18-Tetraoxa-1,10-diazabicyclo[8.5.5]tricosane (Kryptofix® 211).

The optimal reaction temperature can be adjusted to take intoconsideration the reactivity of the leaving group and the boiling pointof the solvent used in the reaction. In the absence of solvent, thereaction can be conducted at temperatures up to about 200° C. Therelatively high reaction temperature minimizes the reaction time.Alternatively, a reaction temperature of about 120° C. is optimal forcertain substrates when acetonitrile is used as the solvent. Thefluorination reaction can be carried out at lower reaction temperatures.Lower reaction temperatures can be beneficial for substrates that maydecompose at elevated temperatures. However, the reaction must beconducted over a longer time period to reach completion when thereaction is performed at a lower temperature. In certain embodiments,the reaction is conducted a temperature of at least about 25° C., 40°C., 50° C., or 75° C. In a more preferred embodiment, the reaction thereaction is conducted a temperature of at least about 100° C., 110° C.,120° C., or 140° C. In certain embodiments, the reaction is conducted ata temperature of at least about 180° C., 200° C., or 220° C. Generally,reactions conducted near about 200° C. are performed without solvent.

The leaving group can be any chemical fragment that is capable of beingdisplaced by the halogen nucleophile. In certain embodiments, theleaving group is an acetate, sulfonate, phosphate, halogen, nitro group,and the like. In a preferred embodiment, the leaving group is amesylate, trifluoromethanesulfonate, or nitro group. The leaving groupmay be attached to a primary or second carbon atom of an alkyl orcycloalkyl group. In addition, the halogenation reaction of theinvention also works for leaving groups that are attached to an aromaticring. In a preferred embodiment, the aromatic ring is a benzene ring andthe leaving group is a nitro group.

The halogenation reaction of the invention works best when the reactionconditions are anhydrous. In certain embodiments, the reaction isconducted in the presence of less than about 5%, 3%, 2%, 1%, 0.5%, or0.1% water. In a preferred embodiment, the reaction conditions areanhydrous. It is important that any water is substantially removed fromsolvents that are used. In addition, it is important that the potassiumfluoride is anhydrous. In certain embodiments, the potassium fluoridecontains less than about 3%, 2%, 1%, 0.5%, or 0.1% water by weight. In apreferred embodiment, the potassium fluoride contains less than about 1%water by weight.

The halogenation reaction of the invention can be performed usinghalogen sources other than potassium fluoride. For example, thepotassium cation can be substituted by a lithium, sodium, cesium, orrubidium cation. In addition, the potassium cation can be substituted bya positively charged transition metal, including a lanthanide oractinide. In certain embodiments, the potassium cation can be replacedby a tetralkylammonium cation, e.g., tetrabutyl ammonium. Thehalogenation reaction of the invention can be used to introduce isotopesof fluoride, such as ¹⁸F. In certain embodiments, the halogenationreaction of the invention can be used to introduce isotopes of iodide,bromine or chlorine. In a certain instances, the iodide is aradioisotope, e.g., ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. In certain instances, thechloride is a radioisotope, e.g., ³⁶Cl. In certain instances, thebromine is a radioisotope, e.g., ⁷⁷Br, ⁸⁰Br or ⁸²Br.

In certain embodiments, the reaction may proceed more quickly in thepresence of a transition metal. Certain transition metals are known tocomplex to aromatic carbon atoms, thereby rendering the carbon atomsmore susceptible to attack by a nucleophile. Certain transition metalshave energetically accessible d-orbitals, which are only partiallyfilled with electrons. The number and shape of these orbitals contributeto the large number of reaction pathways that are made possible by thesecatalysts. Metal-activated arenes undergo nucleophilic reactions (FIG.1). The metal acts as a strong electron-withdrawing group often comparedto a nitro group, thus arenes can accept electron density from incomingnucleophiles.

The radiofluorination reaction is amenable to a wide variety ofcompounds including sulfonate, nitro, acetate or halogen derivates ofalkyl, cycloalkyl, aryl, heteroaryl, aralkyl or alkenyl compounds. Incertain preferred embodiments, the radiofluorination substrate is anitroaromatic compound, alkyl mesylate, or cycloalkylmesylate. Alkenylhalides and alkenyl acetates would also be amenable to the reactionconditions. The following prophetic examples illustrate that theradiofluorination reaction of the invention could be used to prepareF-18 labeled alkenyl compounds.

Another aspect of the present invention relates to a method of preparingaryl halides by reacting a triazine with sodium iodide andchlorotrimethylsilane. In a preferred embodiment, the iodide isradioactive. In certain embodiments, the iodide is ¹²³I, ¹²⁴I, ¹²⁵I or¹³¹I. In a preferred embodiment, the iodide is ¹²⁵I. The reaction can beconducted in the presence or absence of solvent. For reactions conductedin the presence of the solvent, the reaction should be amenable to mostorganic solvents which do not have a hydroxyl group that might reactwith the substrates of the reaction. A representative selection ofsuitable solvents includes acetonitrile, dimethylacetamide,dimethylformamide, dimethylsulfoxide, dioxane, benzene, toluene, xylene,ethylbenzene diglyme, dimethoxyethane (glyme), diethylene glycol dibutylether, triethylene glycol dimethyl ether, tetraethylene glycol dimethylether, polyethylene glycol dimethyl ethers, diethylene glycol dibutylether, polyethylene glycol dibutyl ethers, heptane, octane, butylacetateand the like. The ideal solvent for a particular reaction can bedetermined by one of ordinary skill in the art taking into considerationthe preferred temperature of the reaction, the boiling point of thesolvent, and the solubilities of the substrates in the solvent.

The optimal reaction temperature can be adjusted to take intoconsideration the thermal sensitivity of the substrate and the boilingpoint of the solvent used in the reaction. For example, a reactiontemperature of about 120° C. is optimal for certain substrates whenacetonitrile is used as the solvent. The iodination reaction can becarried out at lower reaction temperatures. Lower reaction temperaturescan be beneficial for substrates that may decompose at elevatedtemperatures. However, the reaction will generally require more time toreach completion when the reaction is performed at a lower temperature.In certain embodiments, the reaction is conducted a temperature of atleast about 25° C., 40° C., 50° C., or 75° C. In a more preferredembodiment, the reaction the reaction is conducted a temperature of atleast about 100° C., 110° C., 120° C., or 140° C. However in certainembodiments, the reaction may be conducted at a temperature of at leastabout 150° C. or 180° C.

The halogenation reaction of the invention can be performed usinghalogen sources other than sodium iodide. For example, the sodium cationcan be replaced by a lithium, potassium, cesium, or rubidium cation. Inaddition, the sodium cation can be replaced by a positively chargedtransition metal, including a lanthanide or actinide. The halogenationreaction of the invention can also be used to introduce isotopes ofiodide, such as ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. In certain embodiments, thehalogenation reaction of the invention can be used to introducefluoride, chloride or bromide. In a certain embodiments, the fluoride isa radioisotope, e.g., ¹⁸F. In certain instances, the chloride is aradioisotope, e.g., ³⁶Cl. In certain instances, the bromine is aradioisotope, e.g., ⁷⁷Br, ⁸⁰Br or ⁸²Br.

The chlorosilane can be any trialkylchlorosilane, triarylchlorosilane,triaralkylchlorolsilane, or a chlorosilane that has 1-2 alkyl groups and1-2 aryl groups, such that the sum of the number of alkyl and arylgroups is equal to three. In certain embodiments, the chlorosilane istert-butyl dimethylchlorosilane, triethylchlorosilane,triethylchlorosilane, or trimethylchlorosilane,diphenylchloromethylsilane. In a preferred embodiment, the chlorosilaneis trimethylchlorosilane.

The present invention provides a method for the introduction offluoride-18 to many organic molecules using a catalytic exchangereaction. One advantage of this method is its simplicity and itspotential to allow the radiolabeling of many biologically activemolecules with a simple form of fluoride-18 produced on a routine basisin many facilities. This method will allow widespread production and useof many new and valuable radiopharmaceutical. The radiofluorinationmethod of the invention is useful for installation of fluoride-18 andother halogens on single, double or aromatic bonds, i.e., sp³-hybridizedand sp²-hybridized carbons.

Biodistribution Analysis

Rats

CD Fischer rats (175-200 g) will be anesthetized with ether, and 0.2 mL(20-40 mCi) of the F-18-labeled compound will be injected via the tailvein. Groups of six rats each will be sacrificed by ether asphyxiationat 5, 30 and 60 min post administration. The appropriate organs will beexcised, blotted dry, weighed, and assayed for radioactivity in aNaI(Tl) gamma well scintillation counter. Blood will be obtained from avein in the thoracic cavity and assayed for radioactivity.

Monkeys

The animals will be positioned in the micro PET tomographic camera, and2 to 5 mCi of the F-18-methylated phenylpiperazinium derivative in 2.5mL of 0.9% saline will be injected iv over several seconds. PET datawill be collected over a 1-h period and 10-sec integration exposureswill be used for the first 10 min and 1-min exposures thereafter. Thedata will be corrected for scatter, accidental coincidences,self-absorption and detector uniformity variations. Quantitativetomographic constructions will be then computed. Time-activity curvesfor regions of interest will be collected.

The parameters of interest in the evaluation of these radiochemicals aspotential myocardial imaging agents include uptake in the heart,selectivity for the heart compared to surrounding tissues such as theliver, lungs, and blood, as well as retention of activity in the heart.

According to the present invention,[F-18]-1-methyl-1-(fluoroalkyl)-4-phenylpiperazinium derivatives can bereadily prepared and they possess marked myocardial uptake andselectivity. The radiolabeled compounds will localize rapidly in the ratmyocardium to give both high uptake, good target to non-targetselectivity with significant retention. The tissue distribution will becompared to that of the ¹²⁵I-labeled compounds which were previouslyevaluated. Further preclinical studies will compare the distribution ofthese agents to microspheres and other perfusion markers.

Method for Validating an Agent as a Blood-Flow Agent by PET Imaging andMicrosphere Injection

Animals (pigs or rats) are prepared according to an accepted protocol,in brief, by inserting a catheter in the carotid artery and the femoralvein. The animals are positioned in the PET camera.

Two sets with different tag microspheres and blood flow agent areinjected in a two-part experiment as follows. In both parts,microspheres are injected in one line (arterial) and blood collectedfrom the other using pumps with specific flow rates. 1. The firstinjection is at the normal blood flow state of the animal. A [F-18]blood flow agent and the first tag microspheres are injected. Bloodsampling with a known flow rate are drawn and an imaging schedule withPET is performed (see below image collection schedule) and 2. The secondtag microspheres and the same [F-18] blood flow agent are injected afterincreasing the blood flow by an adenosine bolus injection. The blooddrawing and imaging schedule is repeated as above. At the end of thesecond imaging procedure the animal is sacrificed, the heart is removedand the blood and heart tissue are counted for microspheresconcentrations. Blood flow is calculated counted. (Carter, E., J. Appl.Physiol., 1988). The increase in blood flow measured by the microspheres(gold standard) is compared to the one obtained by imaging. In the humanmicrosphere imaging measurements are not allowed.

Methods for Monitoring Blood Flow and Membrane Transport

Imaging Schedule: For purposes of PET imaging, the animals arepositioned in the positron camera using a plastic-imaging cradle. Priorto imaging, transmission data will be acquired with rotating pin sourcecontaining 68Ga for subsequent attenuation correction of PET scans.After injection with approximately 2-10 mCi of the [18F] blood flowagent, arterial blood samples will be obtained at 1, 3, 6, 9, 12, 15,20, 25, 30, and 60 min. Sequential imaging collections of 30-60 secondframes are obtained and the pharmacokinetics of the blood flow agent inthe heart is determined by plotting heart activity as a function oftime.

The procedure for monitoring membrane transport is analagous to thatdescribed for monitoring blood flow.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “leaving group” refers to a functionality which upon bondcleavage departs with an electron pair. In general, good leaving groupsare those moieties which are expelled from the substrate as weak bases.For example, sulfates, sulfonates, chloride, bromide, iodide, phosphatesand the like are good leaving groups. In addition, some moieties may begood leaving groups when protonated or complexed with a Lewis acid. Forexample, alkoxide ions are generally poor leaving groups, but alcoholsare good leaving groups. It should be noted that ring strain may, insome cases, allow a rather poor leaving group to be expelled, as in thecase of epoxides, aziridines, and the like.

The term “crown ether” refers to a cyclic molecule in which ether groups(i.e., polyethers) are connected by dimethylene linkages.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” areart-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on 560 of “Advancedinorganic Chemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In other embodiments, R50 and R51(and optionally R52) each independently represent a hydrogen, an alkyl,an alkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S— alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carboxyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 andR56 represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61 or apharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the formula representsan “ester”. Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the formula represents a “carboxylic acid”. Where X50is an oxygen, and R56 is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the formula represents a“thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the formula represents a “thiolformate.” On the other band,where X50 is a bond, and R55 is not hydrogen, the above formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above formula represents an “aldehyde” group.

The term “carbamoyl” refers to —O(C═O)NRR′, where R and R′ areindependently H, aliphatic groups, aryl groups or heteroaryl groups.

The term “oxo” refers to a carbonyl oxygen (═O).

The terms “oxime” and “oxime ether” are art-recognized and refer tomoieties that may be represented by the general formula:

wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61. The moiety is an “oxime” when R is H; and itis an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R58 is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The term “selenoalkyl” is art-recognized and refers to an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R61, m andR61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991). Protected forms of the inventive compounds are included withinthe scope of this invention.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Compounds of the Invention

One aspect of the current invention relates to F-18 cationic andcationic lipophilic compounds useful as blood flow markers formyocardial PET imaging. In certain embodiments, the compounds arecharged phenylpiperazine and tetraphenylphosphonium compounds. Thecompounds of the invention have superior properties to other imagingagents because F-18 is a better radiolabel than Tc-99m (PET Vs. SPECT).For example, the high resolution and shorter half-life of F-18 render ita superior agent for imaging. In addition, the compounds of theinvention should exhibit: 1) behavior similar to that of microspheres,as opposed to free diffusion in and out of the cell; and 2)receptor-binding advantages due to the piperazine core. Moreover, F-18tetraphenyl phosphonium compounds are potentially useful for imagingbrain tumors. The superior technical attributes of these compoundsrelate in part to the fact that such PET imaging agents are moresuitable for regional quantitation of a measured physiological parameterdue to the simultaneous coincidence detection (180°) of the positroninhalation. This in turn increases the accuracy and sensitivity toin-depth resolution. In addition, tetraphenylphosphonium agents can beused to image tumors, although not necessarily brain tumors.Tetraphenylphosphonium agents will concentrate in tumors that haveenhanced negative charge on cell membrane and mitochondria, such asbreast carcinoma. Finally, the compounds of the present invention may beused to assess qualitatively and quantitatively blood flow and membranetransport in a mammal.

One aspect of the present invention relates to a compound represented byformula I:

wherein

R¹ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, or—CO₂R⁶;

R² represents independently for each occurrence H, alkyl, halogen,hydroxyl, amino, aminoalkyl, or alkoxyl;

R³ represents independently for each occurrence H, alkyl, or halogen;

R⁴ is alkyl or aralkyl;

R⁵ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl,fluorosubstituted aryl, fluorosubstituted aralkyl, or fluorosubstitutedalkenyl; and said fluoro substituent comprises ¹⁸F;

X is an anion that has an overall charge of −1 and

R⁶ is H, alkyl, aryl, or aralkyl.

In certain embodiments, the present invention relates to compound I,wherein said compound has a radioactivity of greater than or equal toabout 1 Curie/mmol.

In certain embodiments, the present invention relates to compound I,wherein said compound has a radioactivity of greater than or equal toabout 5 Curie/mmol.

In certain embodiments, the present invention relates to compound I,wherein said compound has a radioactivity of greater than or equal toabout 10 Curie/mmol.

In certain embodiments, the present invention relates to compound I,wherein said compound has a radioactivity of greater than or equal toabout 100 Curie/mmol.

In certain embodiments, the present invention relates to compound I,wherein said compound has a radioactivity of greater than or equal toabout 1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound I,wherein R¹ is H, alkyl, cycloalkyl, or aryl.

In certain embodiments, the present invention relates to compound I,wherein R¹ is H.

In certain embodiments, the present invention relates to compound I,wherein R¹ is aryl.

In certain embodiments, the present invention relates to compound I,wherein R¹ is a phenyl group.

In certain embodiments, the present invention relates to compound I,wherein R² and R³ represent independently for each occurrence H oralkyl.

In certain embodiments, the present invention relates to compound I,wherein R² and R³ represent independently for each occurrence H.

In certain embodiments, the present invention relates to compound I,wherein R⁴ is alkyl.

In certain embodiments, the present invention relates to compound I,wherein R⁴ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, or pentyl.

In certain embodiments, the present invention relates to compound I,wherein R⁴ is methyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl, orfluorosubstituted aryl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted cycloalkyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted cyclopropyl, fluorosubstitutedcyclobutyl, fluorosubstituted cyclopentyl, fluorosubstituted cyclohexyl,fluorosubstituted cycloheptyl, or fluorosubstituted cyclooctyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted cyclobutyl or fluorosubstitutedcyclohexyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is 2-fluorocyclobutyl or 4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted aryl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is fluorosubstituted phenyl.

In certain embodiments, the present invention relates to compound I,wherein R⁵ is 4-fluorophenyl.

In certain embodiments, the present invention relates to compound I,wherein X is halide, acetate, nitrate, sulfonate, PO₄M₂, SO₄M, valerate,oleate, palmitate, stearate, laurate, or benzoate; wherein M is alkalimetal.

In certain embodiments, the present invention relates to compound I,wherein X is halide, acetate, or nitrate.

In certain embodiments, the present invention relates to compound I,wherein X is nitrate.

In certain embodiments, the present invention relates to compound I,wherein X is halide.

In certain embodiments, the present invention relates to compound I,wherein X is chloride or iodide.

In certain embodiments, the present invention relates to compound I,wherein R¹, R², and R³ represent independently for each occurrence H; R⁴is methyl; and R⁵ is fluorosubstituted aryl.

In certain embodiments, the present invention relates to compound I,wherein R¹, R², and R³ represent independently for each occurrence H; R⁴is methyl; and R⁵ is 4-fluorophenyl.

In certain embodiments, the present invention relates to compound I,wherein R¹, R², and R³ represent independently for each occurrence H; R⁴is methyl; R⁵ is 4-fluorophenyl; and X is chloride.

In certain embodiments, the present invention relates to compound I,wherein R¹ is phenyl, R² and R³ represent independently for eachoccurrence H, R⁴ is methyl, and R⁵ is fluorosubstituted cycloalkyl.

In certain embodiments, the present invention relates to compound I,wherein R¹ is phenyl, R² and R³ represent independently for eachoccurrence H, R⁴ is methyl, and R⁵ is 2-fluorocyclobutyl or4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound I,wherein R¹ is phenyl, R² and R³ represent independently for eachoccurrence H, R⁴ is methyl, X is iodide, and R⁵ is 2-fluorocyclobutyl or4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound I,wherein the fluoro substituent of R⁵ comprises ¹⁸F at natural abundance.

Another aspect of the present invention relates to a compoundrepresented by formula II:

wherein

R¹ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl,fluorosubstituted aryl, fluorosubstituted aralkyl, or fluorosubstitutedalkenyl; wherein said fluoro substituent comprises ¹⁸F;

R² represents independently for each occurrence H, alkyl, halogen,hydroxyl, amino, aminoalkyl, or alkoxyl;

R³ represents independently for each occurrence H, alkyl, or halogen;

R⁴ is alkyl or aralkyl;

R⁵ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, or—CO₂R⁶;

X is an anion that has an overall charge of −1 and

R⁶ is H, alkyl, aryl, or aralkyl.

In certain embodiments, the present invention relates to compound II,wherein said compound has a radioactivity of greater than or equal toabout 1 Curie/mmol.

In certain embodiments, the present invention relates to compound II,wherein said compound has a radioactivity of greater than or equal toabout 5 Curie/mmol.

In certain embodiments, the present invention relates to compound II,wherein said compound has a radioactivity of greater than or equal toabout 10 Curie/mmol.

In certain embodiments, the present invention relates to compound II,wherein said compound has a radioactivity of greater than or equal toabout 100 Curie/mmol.

In certain embodiments, the present invention relates to compound II,wherein said compound has a radioactivity of greater than or equal toabout 1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound II,wherein R⁵ is H, alkyl, cycloalkyl, or aryl.

In certain embodiments, the present invention relates to compound II,wherein R⁵ is H.

In certain embodiments, the present invention relates to compound II,wherein R⁵ is aryl.

In certain embodiments, the present invention relates to compound II,wherein R⁵ is a phenyl group.

In certain embodiments, the present invention relates to compound II,wherein R² and R³ represent independently for each occurrence H oralkyl.

In certain embodiments, the present invention relates to compound II,wherein R² and R³ represent independently for each occurrence H.

In certain embodiments, the present invention relates to compound II,wherein R⁴ is alkyl.

In certain embodiments, the present invention relates to compound II,wherein R⁴ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, or pentyl.

In certain embodiments, the present invention relates to compound II,wherein R⁴ is methyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl, orfluorosubstituted aryl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted cycloalkyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted cyclopropyl, fluorosubstitutedcyclobutyl, fluorosubstituted cyclopentyl, fluorosubstituted cyclohexyl,fluorosubstituted cycloheptyl, or fluorosubstituted cyclooctyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted cyclobutyl or fluorosubstitutedcyclohexyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is 2-fluorocyclobutyl or 4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted aryl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is fluorosubstituted phenyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is 4-fluorophenyl.

In certain embodiments, the present invention relates to compound II,wherein X is halide, acetate, nitrate, sulfonate, PO₄M₂, SO₄M, valerate,oleate, palmitate, stearate, laurate, or benzoate; wherein M is alkalimetal.

In certain embodiments, the present invention relates to compound II,wherein X is halide, acetate, or nitrate.

In certain embodiments, the present invention relates to compound II,wherein X is halide.

In certain embodiments, the present invention relates to compound II,wherein X is nitrate.

Another aspect of the present invention relates to a compoundrepresented by formula III:

wherein

R¹ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, or—CO₂R⁵;

R² represents independently for each occurrence H, alkyl, halogen,hydroxyl, amino, aminoalkyl, or alkoxyl;

R³ represents independently for each occurrence H, alkyl, or halogen;

R⁴ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl,fluorosubstituted aryl, fluorosubstituted aralkyl, or fluorosubstitutedalkenyl; and said fluoro substituent comprises ¹⁸F;

X is an anion that has an overall charge of −1; and

R⁵ is H, alkyl, aryl, or aralkyl.

In certain embodiments, the present invention relates to compound III,wherein said compound has a radioactivity of greater than or equal toabout 1 Curie/mmol.

In certain embodiments, the present invention relates to compound III,wherein said compound has a radioactivity of greater than or equal toabout 5 Curie/mmol.

In certain embodiments, the present invention relates to compound III,wherein said compound has a radioactivity of greater than or equal toabout 10 Curie/mmol.

In certain embodiments, the present invention relates to compound III,wherein said compound has a radioactivity of greater than or equal toabout 100 Curie/mmol.

In certain embodiments, the present invention relates to compound III,wherein said compound has a radioactivity of greater than or equal toabout 1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound III,wherein R¹ is H, alkyl, cycloalkyl, or aryl.

In certain embodiments, the present invention relates to compound III,wherein R¹ is H.

In certain embodiments, the present invention relates to compound III,wherein R¹ is aryl.

In certain embodiments, the present invention relates to compound III,wherein R¹ is a phenyl group.

In certain embodiments, the present invention relates to compound III,wherein R² and R³ represent independently for each occurrence H oralkyl.

In certain embodiments, the present invention relates to compound III,wherein R² and R³ represent independently for each occurrence H.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted alkyl, fluorosubstituted cycloalkyl, orfluorosubstituted aryl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted cycloalkyl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted cyclopropyl, fluorosubstitutedcyclobutyl, fluorosubstituted cyclopentyl, fluorosubstituted cyclohexyl,fluorosubstituted cycloheptyl, or fluorosubstituted cyclooctyl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted cyclobutyl or fluorosubstitutedcyclohexyl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is 2-fluorocyclobutyl or 4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted aryl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is fluorosubstituted phenyl.

In certain embodiments, the present invention relates to compound III,wherein R⁴ is 4-fluorophenyl.

In certain embodiments, the present invention relates to compound III,wherein R¹, R², and R³ represent independently for each occurrence H;and R⁴ is fluorosubstituted aryl.

In certain embodiments, the present invention relates to compound III,wherein R¹, R², and R³ represent independently for each occurrence H;and R⁴ is 4-fluorophenyl.

In certain embodiments, the present invention relates to compound III,wherein R¹ is phenyl, R² and R³ represent independently for eachoccurrence H, and R⁴ is fluorosubstituted cycloalkyl.

In certain embodiments, the present invention relates to compound II,wherein R¹ is phenyl, R² and R³ represent independently for eachoccurrence H, and R⁴ is 2-fluorocyclobutyl or 4-fluorocyclohexyl.

In certain embodiments, the present invention relates to compound III,wherein the fluoro substituent of R⁵ comprises ¹⁸F at natural abundance.

Another aspect of the present invention relates to a compoundrepresented by formula IV:

wherein

R¹ represents independently for each occurrence aryl or heteroaryl;

R² is halogen-substituted alkyl, halogen-substituted cycloalkyl,halogen-substituted aryl, halogen-substituted aralkyl,halogen-substituted alkenyl; wherein said halogen substituent isfluoride that comprises ¹⁸F, or said halogen substituent is iodide thatcomprises ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I; and

X is an anion that has an overall charge of −1.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence aryl.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence optionallysubstituted phenyl.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence phenyl.

In certain embodiments, the present invention relates to compound IV,wherein R² is halogen-substituted cycloalkyl or halogen-substitutedaryl.

In certain embodiments, the present invention relates to compound IV,wherein R² is halogen-substituted aryl.

In certain embodiments, the present invention relates to compound IV,wherein R² is halogen-substituted phenyl.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence phenyl and R² is4-fluorophenyl.

In certain embodiments, the present invention relates to compound IV,wherein X is halide, acetate, nitrate, sulfonate, PO₄M₂, SO₄M, valerate,oleate, palmitate, stearate, laurate, or benzoate; wherein M is alkalimetal.

In certain embodiments, the present invention relates to compound IV,wherein X is halide, acetate, or nitrate.

In certain embodiments, the present invention relates to compound IV,wherein X is nitrate.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence phenyl, R² is4-fluorophenyl, and X is nitrate.

In certain embodiments, the present invention relates to compound IV,wherein R¹ represents independently for each occurrence phenyl, R² is4-iodophenyl, and X is nitrate.

Another aspect of the present invention relates to a compoundrepresented by formula V:

wherein

R¹ represents independently for each occurrence aryl or heteroaryl;

R² is halogen-substituted alkyl, halogen-substituted cycloalkyl,halogen-substituted aryl, halogen-substituted aralkyl,halogen-substituted alkenyl; wherein said halogen substituent isfluoride that comprises ¹⁸F, or said halogen substituent is iodide thatcomprises ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I; and

X is an anion that has an overall charge of −1.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence aryl.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence optionallysubstituted phenyl.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence phenyl.

In certain embodiments, the present invention relates to compound V,wherein R² is halogen-substituted cycloalkyl or halogen-substitutedaryl.

In certain embodiments, the present invention relates to compound V,wherein R² is halogen-substituted aryl.

In certain embodiments, the present invention relates to compound V,wherein R² is halogen-substituted phenyl.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence phenyl and R² is4-fluorophenyl.

In certain embodiments, the present invention relates to compound V,wherein X is halide, acetate, nitrate, sulfonate, PO₄M₂, SO₄M, valerate,oleate, palmitate, stearate, laurate, or benzoate; wherein M is alkalimetal.

In certain embodiments, the present invention relates to compound V,wherein X is halide, acetate, or nitrate.

In certain embodiments, the present invention relates to compound V,wherein X is nitrate.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence phenyl, R² is4-fluorophenyl, and X is nitrate.

In certain embodiments, the present invention relates to compound V,wherein R¹ represents independently for each occurrence phenyl, R² is4-iodophenyl, and X is nitrate.

Another aspect of the present invention relates to a compoundrepresented by formula VI:

wherein

R¹ represents independently for each occurrence aryl or heteroaryl;

R² is halogen-substituted alkyl, halogen-substituted cycloalkyl,halogen-substituted aryl, halogen-substituted aralkyl,halogen-substituted alkenyl; wherein said halogen substituent isfluoride that comprises ¹⁸F, or said halogen substituent is iodide thatcomprises ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I; and

X is an anion that has an overall charge of −1.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is fluoride that comprises ¹⁸F;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²³I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁴I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹²⁵I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about5 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about10 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about100 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein said halogen substituent of R² is iodide that comprises ¹³¹I;and said compound has a radioactivity of greater than or equal to about1,000 Curie/mmol.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence aryl.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence optionallysubstituted phenyl.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence phenyl.

In certain embodiments, the present invention relates to compound VI,wherein R² is halogen-substituted cycloalkyl or halogen-substitutedaryl.

In certain embodiments, the present invention relates to compound VI,wherein R² is halogen-substituted aryl.

In certain embodiments, the present invention relates to compound VI,wherein R² is halogen-substituted phenyl.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence phenyl and R² is4-fluorophenyl.

In certain embodiments, the present invention relates to compound VI,wherein X is halide, acetate, nitrate, sulfonate, PO₄M₂, SO₄M, valerate,oleate, palmitate, stearate, laurate, or benzoate; wherein M is alkalimetal.

In certain embodiments, the present invention relates to compound VI,wherein X is halide, acetate, or nitrate.

In certain embodiments, the present invention relates to compound VI,wherein X is nitrate.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence phenyl, R² is4-fluorophenyl, and X is nitrate.

In certain embodiments, the present invention relates to compound VI,wherein R¹ represents independently for each occurrence phenyl, R² is4-iodophenyl, and X is nitrate.

Another aspect of the present invention relates to a formulation,comprising a compound of formula I, II, III, IV, V, or VI; and apharmaceutically acceptable excipient.

Methods of the Invention

One aspect of the present invention relates to a method of making ahalogenated compound as depicted in Scheme 1:

wherein

A is alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, alkenyl or has theformula a, b, c, or d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl;    -   R³ represents independently for each occurrence H, alkyl, or        halogen;    -   R⁴ represents independently for each occurrence H, alkyl,        halogen, hydroxyl, amino, aminoalkyl, or alkoxyl;    -   R⁵ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,        aralkyl, heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl,        aralkylsulfonyl, or —CO₂R⁵; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate;

X is sulfonate, nitro, acetate, or halogen;

M is an alkali metal or transition metal;

Y is fluoride or iodide;

crown ether is a cyclic molecule in which oxygen atoms are connected byoptionally substituted dimethylene linkages; and

the method is practiced under substantially anhydrous conditions.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is a radioactive fluoride orradioactive iodide.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹⁸F.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²³I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²⁴I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²⁵I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹³¹I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein the method is practiced under anhydrousconditions.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl, cycloalkyl, or aryl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl and said alkyl group issubstituted by X at a primary carbon atom.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl and said alkyl group issubstituted by X at a secondary carbon atom.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cycloalkyl and said alkyl group issubstituted by X at a secondary carbon atom.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cyclobutyl or cyclohexyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula a:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula b:

-   -   wherein    -   R² represents independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl;    -   R³ represents independently for each occurrence H, alkyl, or        halogen;    -   R⁴ represents independently for each occurrence H, alkyl,        halogen, hydroxyl, amino, aminoalkyl, or alkoxyl; and    -   R⁵ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,        aralkyl, heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl,        aralkylsulfonyl, or —CO₂R⁵.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula a:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula a:

-   -   wherein    -   R¹ and R² represent independently for each occurrence optionally        substituted phenyl; and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula b:

-   -   wherein    -   R² represents cycloalkyl;    -   R³ represents H;    -   R⁴ represents H; and    -   R⁵ is H or aryl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is a sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is a sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is sulfonate or nitro.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is methanesulfonate ortrifluoromethanesulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is an alkali metal.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is potassium, sodium, or lithium.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is potassium.

In certain embodiments, the present invention relates to theaforementioned method, wherein said crown ether is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein said crown ether is selected from thegroup consisting of 1,4,10-Trioxa-7,13-diaza-cyclopentadecane(Kryptofixa® 21),4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix®222), 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo[8.8.8.5]tricosane(Kryptofix® 221), and4,7,13,18-Tetraoxa-1,10-diazabicyclo[8.5.5]eicosane (Kryptofix® 211).

In certain embodiments, the present invention relates to theaforementioned method, wherein the reaction temperature is between about50° C. and about 220° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the reaction temperature is between about100° C. and about 200° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the reaction is conducted in the presenceof solvent.

In certain embodiments, the present invention relates to theaforementioned method, wherein the reaction is conducted in the presenceof acetonitrile.

In certain embodiments, the present invention relates to theaforementioned method, wherein said MY is anhydrous.

In certain embodiments, the present invention relates to theaforementioned method, wherein said MY contains less than about 2%water.

In certain embodiments, the present invention relates to theaforementioned method, wherein said MY contains less than about 1%water.

In certain embodiments, the present invention relates to theaforementioned method, wherein said MY contains less than about 0.5%water.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl, X is sulfonate, M ispotassium, Y comprises ¹⁸F, and said crown either is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is alkyl, X is methanesulfonate, M ispotassium, Y comprises ¹⁸F, and said crown either is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cycloalkyl, X is sulfonate, M ispotassium, Y comprises ¹⁸F, and said crown either is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cycloalkyl, X is methanesulfonate, Mis potassium, Y comprises ¹⁸F, and said crown either is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is methanesulfonate, M is potassium, Ycomprises ¹⁸F, crown either is Kryptofix, and A is optionallysubstituted cyclobutyl or optionally substituted cyclohexyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is methanesulfonate, M is potassium, Ycomprises ¹⁸F, crown either is Kryptofix, and A is cyclobutyl orcyclohexyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is aryl, X is nitro, M is potassium, Ycomprises ¹⁸F, and said crown either is Kryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is optionally substituted phenyl, X isnitro, M is potassium, Y comprises ¹⁸F, and said crown either isKryptofix.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is cycloalkyl, X is methanesulfonate, Mis potassium, Y comprises ¹⁸F, crown either is Kryptofix, and thereaction temperature is between about 100° C. and about 200° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein A is aryl, X is nitro, M is potassium, Ycomprises ¹⁸F, and said crown either is Kryptofix, and the reactiontemperature is between about 100° C. and about 200° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is nitro, M is potassium, Y comprises¹⁸F, and said crown either is Kryptofix, and the reaction temperature isbetween about 100° C. and about 200° C., and A has the formula a:

-   -   wherein    -   R¹ and R² represent independently for each occurrence optionally        substituted phenyl; and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is methanesulfonate ortrifluoromethanesulfonate; M is potassium; said crown ether isKryptofix; Y comprises ¹⁸F; and A has the formula b:

wherein

R² represents cycloalkyl;

R³ represents independently for each occurrence H;

R⁴ represents independently for each occurrence H; and

R⁵ is H or aryl.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is nitro, M is potassium, Y comprises¹⁸F, and said crown either is Kryptofix, and the reaction temperature isbetween about 100° C. and about 200° C., and A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is nitrate.

In certain embodiments, the present invention relates to theaforementioned method, wherein X is nitro, M is potassium, Y comprises¹⁸F, and said crown either is Kryptofix, and the reaction temperature isbetween about 100° C. and about 200° C., and A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is a sulfonate.

Another aspect of the present invention relates to a method of making ahalogenated compound as depicted in Scheme 2:

wherein

R¹ and R² represent independently for each occurrence alkyl, aryl,aralkyl, or R¹ and R² taken together form a cycloalkyl group;

R is alkenyl, aryl, heteroaryl, or has the formula a, b, c, or d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl;    -   R³ represents independently for each occurrence H, alkyl, or        halogen;    -   R⁴ represents independently for each occurrence H, alkyl,        halogen, hydroxyl, amino, aminoalkyl, or alkoxyl;

R⁵ is H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, acyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, or—CO₂R⁵; and

-   -   Z is halide, nitrate, acetate, benzoate, or sulfonate;

M is an alkali metal, transition metal, or tetralkylammonium salt;

Y is fluoride or iodide; and

R³ represents independently for each occurrence alkyl, aryl, or aralkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹⁸F.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is fluoride that comprises ¹⁸F; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²³I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²³I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²⁴I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁴I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹²⁵I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹²⁵I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y comprises ¹³¹I.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 5Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 10Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 100Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is iodide that comprises ¹³¹I; and theradioactivity of MY, A-Y or both is greater than or equal to about 1,000Curie/mmol.

In certain embodiments, the present invention relates to theaforementioned method, wherein the method is practiced under anhydrousconditions.

In certain embodiments, the present invention relates to theaforementioned method, wherein R is aryl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R has the formula a:

-   -   wherein    -   R⁴ and R⁵ represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein R has the formula a:

-   -   wherein    -   R⁴ and R⁵ represent independently for each occurrence aryl; and    -   Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence alkyl,        cycloalkyl, aryl, or heteroaryl; and    -   Z is halide, nitrate, acetate, benzoate, or sulfonate.

In certain embodiments, the present invention relates to theaforementioned method, wherein A has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and

Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R¹ and R² are taken together to form acycloalkyl group.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is an alkali metal.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is potassium, sodium, or lithium.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium.

In certain embodiments, the present invention relates to theaforementioned method, wherein R³ represents independently for eachoccurrence alkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R³ represents independently for eachoccurrence methyl, ethyl, propyl, isopropyl, or butyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R³ represents independently for eachoccurrence methyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cycloalkyl group, and R has the formula a:

-   -   wherein    -   R⁴ and R⁵ represent independently for each occurrence aryl; and    -   Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cyclohexyl group, and R has the formula a:

-   -   wherein    -   R⁴ and R⁵ represent independently for each occurrence optionally        substituted phenyl; and    -   Z is iodide.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cyclohexyl group, and R has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cyclohexyl group, and R has the formula c:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is iodide.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cyclohexyl group, and R has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence aryl; and    -   Z is halide.

In certain embodiments, the present invention relates to theaforementioned method, wherein M is sodium, Y comprises Iodide-125, R³represents independently for each occurrence methyl, R¹ and R² are takentogether to form a cyclohexyl group, and R has the formula d:

-   -   wherein    -   R¹ and R² represent independently for each occurrence phenyl;        and    -   Z is iodide.

Another aspect of the present invention relates to a method of obtaininga positron emission image of a portion of a mammal, comprising the stepsof:

administering to a mammal a compound of formula I, II, III, IV, V, or VIand acquiring a positron emission spectrum of a portion of said mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaVI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human, mouse, rat, dog,feline, monkey, guinea pig, or rabbit.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human.

Another aspect of the present invention relates to a method of measuringblood flow in the heart of a mammal, comprising the steps of:

administering to a mammal a compound of formula I, II, III, IV, V, VIand acquiring a positron emission spectrum of a portion of said mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaVI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human, mouse, rat, dog,feline, monkey, guinea pig, or rabbit.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human.

Another aspect of the present invention relates to a method of measuringmembrane transport in a mammal, comprising the steps of:

administering to a mammal a compound of formula I, II, III, IV, V, or VIand acquiring a positron emission spectrum of a portion of said mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIII.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaIV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaV.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is represented by formulaVI.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human, mouse, rat, dog,feline, monkey, guinea pig, or rabbit.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” O and B books, Corvallis, Ore., U.S.A., 1977).

Micelles

Recently, the pharmaceutical industry introduced microemulsificationtechnology to improve bioavailability of some lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other things, microenmulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom a compound of the present invention and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastrointestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Particularly preferred amphiphilic carriers are saturated andmonounsaturated polyethyleneglycolyzed fatty acid glycerides, such asthose obtained from fully or partially hydrogenated various vegetableoils. Such oils may advantageously consist of tri-. di- and mono-fattyacid glycerides and di- and mono-polyethyleneglycol esters of thecorresponding fatty acids, with a particularly preferred fatty acidcomposition including capric acid 4-10, capric acid 3-9, lauric acid40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%.Another useful class of amphiphilic carriers includes partiallyesterified sorbitan and/or sorbitol, with saturated or mono-unsaturatedfatty acids (SPAN-series) or corresponding ethoxylated analogs(TWEEN-series).

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-scries, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Polymers

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter .alpha., .beta. or.gamma., respectively. Cyclodextrins with fewer than six glucose unitsare not known to exist. The glucose units are linked byalpha-1,4-glucosidic bonds. As a consequence of the chair conformationof the sugar units, all secondary hydroxyl groups (at C-2, C-3) arelocated on one side of the ring, while all the primary hydroxyl groupsat C-6 are situated on the other side. As a result, the external facesare hydrophilic, making the cyclodextrins water-soluble. In contrast,the cavities of the cyclodextrins are hydrophobic, since they are linedby the hydrogen of atoms C-3 and C-5, and by ether-like oxygens. Thesematrices allow complexation with a variety of relatively hydrophobiccompounds, including, for instance, steroid compounds such as17.beta.-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org.Cult. 38:1-3-113 (1994)). The complexation takes place by Van der Waalsinteractions and by hydrogen bond formation. For a general review of thechemistry of cyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl.,33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosuliphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C.sub.14 to about C.sub.20).Polymer-derivatized lipids such as PEG-lipids may also be utilized formicelle formation as they will act to inhibit micelle/membrane fusion,and as the addition of a polymer to surfactant molecules decreases theCMC of the surfactant and aids in micelle formation. Preferred aresurfactants with CMCs in the micromolar range; higher CMC surfactantsmay be utilized to prepare micelles entrapped within liposomes of thepresent invention, however, micelle surfactant monomers could affectliposome bilayer stability and would be a factor in designing a liposomeof a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

Release Modifiers

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

Combinatorial Libraries

The subject compounds may be synthesized using the methods ofcombinatorial synthesis described in this section. Combinatoriallibraries of the compounds may be used for the screening ofpharmaceutical, agrochemical or other biological or medically-relatedactivity or material-related qualities. A combinatorial library for thepurposes of the present invention is a mixture of chemically relatedcompounds which may be screened together for a desired property; saidlibraries may be in solution or covalently linked to a solid support.The preparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes which need tobe carried out. Screening for the appropriate biological,pharmaceutical, agrochemical or physical property may be done byconventional methods.

Diversity in a library can be created at a variety of different levels.For instance, the substrate aryl groups used in a combinatorial approachcan be diverse in terms of the core aryl moiety, e.g., a variegation interms of the ring structure, and/or can be varied with respect to theother substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93/09668 and WO91/07087; and the Lerner et al. PCT publicationWO93/20242). Accordingly, a variety of libraries on the order of about16 to 1,000,000 or more diversomers can be synthesized and screened fora particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group, e.g.,located at one of the positions of substrate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. In one embodiment, which is particularly suitable for discoveringenzyme inhibitors, the beads can be dispersed on the surface of apermeable membrane, and the diversomers released from the beads by lysisof the bead linker. The diversomer from each bead will diffuse acrossthe membrane to an assay zone, where it will interact with an enzymeassay. Detailed descriptions of a number of combinatorial methodologiesare provided below.

A) Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), e.g.,which can be used to characterize sub-femtomolar amounts of a compound,and to directly determine the chemical constitution of a compoundselected from a combinatorial library. For instance, where the libraryis provided on an insoluble support matrix, discrete populations ofcompounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy is introduced intothe library, the beads are divided into separate groups equal to thenumber of different substituents to be added at a particular position inthe library, the different substituents coupled in separate reactions,and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-men, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality in synthesisis achieved by employing acid-labile protection for the coding strandand base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound (Ptek et al. (1991) Tetrahedron Lett32:3891-3894). In another embodiment, the cleavable linker can be placedso that the test compound can be selectively separated from the bead,leaving the code behind. This last construct is particularly valuablebecause it permits screening of the test compound without potentialinterference of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-Sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequenceable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tagsare haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem59:4723-4724). This orthogonal attachment strategy permits the selectivedetachment of library members for assay in solution and subsequentdecoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Synthesis of Precursor 4-nitrophenyltriphenylammonium nitrate

Triphenylamine and 1,4-dinitrobenzene (1 eq) are heated in DMSO underpressure (stainless steel reactor) at 200° C. for 6 h. The ammonium saltis purified by recrystallization.

Example 2 Synthesis of [¹⁸F]FTPN

[¹⁸F]FTPN is prepared from 4-nitrophenyltriphenylammonium nitrate and[¹⁸F]fluoride by nucleophilic aromatic substitution. A Wheaton 5-mLreaction vial containing fluorine-18 in 0.5 mL of ¹⁸O-enriched water and100 μL of ammonia hydroxide is heated at 120° C. and water is evaporatedto dryness with the aid of a nitrogen gas stream. The contents are driedby the addition of 1 mL of acetonitrile followed by evaporation ofsolvent using a nitrogen flow. This process is repeated three times. A100 μL solution of the labeling precursor in dry DMSO was added to thevial containing the activity. The sealed reaction vial is heated at 200°C. for 10 min, cooled to 25° C., and the solution is diluted with 0.5 mlof 0.1M Ca(NO₃)₂. The solution is loaded onto a C-18 SepPak (Waters,Milford, Mass.) and eluted with water (10 mL) to remove unreactedfluoride and DMSO. The crude product is eluted from the SepPak withmethylene chloride (1 mL) and dried with sodium sulfate. The solution isthen loaded onto a HPLC column (Waters: C-18 7.8×300 mm column, flow: 6mL/min; eluent: acetonitrile/water (50/50) and purified. Solvent isremoved by roto-evaporation and [¹⁸F]FTPN is dissolved in saline andfiltered (0.22 μm, Millipore:Millex-GV).

Example 3 Synthesis of Precursor 4-N,N,N-trimethylammoniodiphenylsulfone

4-Dimethylaminophenyl sulfone is prepared using the method ofBaarschers, W. H [Can. J. Chem. (1976), 54(19): 3056]. Phenyl benzenesulfonate is added to an ether solution of dimethylanilinyllithiumprepared from dimethylaniline and n-butyllithium at 25° C.4-Dimethylaminophenyl sulfone in methylene chloride at 25° C. is treatedwith 1.1 equivalence of methyl trifluoromethanesulfonate. After stirringfor 16 hr, ether is added and the precipitate is removed by filtration.

Example 4 Synthetic Method A for Preparing [¹⁸F]FTPS

A Wheaton 5-mL reaction vial containing fluorine-18 in 1-0.5 mL of¹⁸O-enriched water, Kryptofix-2.2.2. (8 mg), and potassium carbonate (4mg) was heated at 120° C. and solvent was evaporated with the aid ofnitrogen gas. The K¹⁸F/Kryptofix complex was dried three times by theaddition of 1 mL of acetonitrile followed by evaporation of the solventusing a nitrogen flow. A solution of 3 mg of 4-nitrodiphenyl sulfone in0.4 mL of acetonitrile was added to the vial and fluorination wasperformed at 140° C. (sealed vial) for 10 min. The solution was loadedonto a silica gel Sep-Pak (Waters, Milford, Mass.) and the activity waseluted with 3 mL of methylene chloride. The eluent containing thelabeled sulfone was placed in a 5-mL vial and solvent was removed by anitrogen stream. A 1M hexane solution of diisobutylaluminum hydride (0.2mL) was added to the reaction vial at 25° C. After 1 min, 10% HC (0.5mL) was added and the labeled sulfide was exacted with methylenechloride (2 ml). The solution containing the activity was then passedthrough a silica gel Sep-Pak and collected in a 5-mL vial.3-Chloroperoxybenzoic acid (50 mg) was added to the vial at 25° C. After10 min, the reaction mixture was passed through a silica gel Sep-Pak andcollected in a 5 mL vial, To the solution of labeled phenyl sulfoxide at−70 C was added triethylamine (50 μL) and trimethylsilyltriflate (50μL). The mixture was then stirred at 5-0° C. for 1 min. The mixture wascooled again to −70° C. and 1.8 M phenyl magnesium chloride in THF (100μL) was added. The mixture was warmed to 5-0° C. (ice-bath) and waterwas added to the reaction mixture to decompose the excess Grignardreagent methylene chloride was removed with a nitrogen stream andreplaced with acetonitrile (1 mL). The solution was then loaded onto aHPLC column (Waters: C-18 7.8×300 mm column, flow: 6 mL/min; eluent:acetonitrile/water (50/50) and purified. Solvent was removed byroto-evaporation and [¹⁸F]FTPS was dissolved in saline and filtered(0.22 μm, Millipore:Millex-GV).

Example 5 Synthesis of Precursors 4-N,N,N-trimethylammoniodiphenylsulfonium trifluoromethanesulfonic acid and 4-nitrophenylsulfoniumtrifluoromethanesulfonic acid

To a solution of phenyl sulfoxide and triethylamine (2 eq) in methylenechloride cooled to −70° C. is added dropwise trimethylsilyltriflate (2eq) keeping the temperature below −60° C. The mixture is then stirred at5-0° C. for 10 min. The mixture is cooled again to −70° C. and theGrignard reagent (4 eq), prepared from either4-bromo-N,N-dimethylaniline or 1-bromo-4-nitrobenzene and magnesium inTHE, is added dropwise to the reaction mixture while controlling thetemperature so as not to exceed −60° C. Thereafter, the mixture isstirred for a further 60 min at 5-0° C. Water is added dropwise to thereaction mixture to decompose the excess Grignard reagent and theinorganic salts are removed by filtration. The organic layer is washedwith water (3×) and solvent is evaporated in vacuo. The oil obtained ischromatographed on silica get to afford the substituted phenylsulfoniumtrifluoromethanesulfonic acid.

A mixture of 4-dimethylaminophenylsulfonium trifluoromethanesulfonicacid and dimethyl sulfate (0.5 g, 4 mmol) in toluene (50 mL) is heatedat 100° C. for 4 hr. Solvent is decanted off and the oil ischromatographed on silica gel using methanol and 2% TFA to give4-N,N,N-trimethylammoniodiphenyl sulfonium trifluoromethanesulfonicacid.

Example 6 Synthetic Method B for Preparing [¹⁸F]FTPS

[¹⁸F]FTPS is prepared from either 4-N,N,N-trimethylammoniodiphenylsulfonium trifluoromethanesulfonic acid or 4-nitrophenylsulfoniumtrifluoromethanesulfonic acid and [¹⁸F]fluoride by nucleophilic aromaticsubstitution. A Wheaton 5-mL reaction vial containing fluorine-18 in 0.5mL of ¹⁸O-enriched water and 100 μL of ammonium hydroxide is heated at120° C. and water is evaporated to dryness with the aid of a nitrogengas stream. The contents are dried by the addition of 1 mL ofacetonitrile followed by evaporation of solvent using a nitrogen flow.This process is repeated three times. A 100 μL solution of the labelingprecursor in dry DMSO was added to the vial containing the activity. Thesealed reaction vial is heated at 160° C. for 10 min, cooled to 25° C.,and the solution is diluted with 0.5 ml of 0.1M Ca(NO₃)₂. The solutionis loaded onto a C-18 SepPak (Waters, Milford, Mass.) and eluted withwater (10 mL) to remove unreacted fluoride and DMSO. The crude productis eluted from the SepPak with acetonitrile (1 mL) and dried with sodiumsulfate. The solution is then loaded onto a HPLC column (Waters: C-187.8×300 mm column, flow: 6 mL/min; eluent: acetonitrile/water (50/50)and purified. Solvent is removed by roto-evaporation and [¹⁸F]FTPS isdissolved in saline and filtered (0.22 μm, Millipore:Millex-GV).

Example 7 Alternative Purification Method: Nitro Group Reduction

After heating the labeling nitro precursor in DMSO (0.2 mL), thereaction mixture is diluted with 0.1M Ca(NO₃)₂ (0.5 mL) and loaded ontoa C-18 Sep-Pak. The Sep-Pak is washed with water (10 mL) and the labeledcompound is eluded with ethanol (1 mL) into a 5 mL vial containing SnCl₂dihydrate (0.1 mmol). The reaction vial is heated at 70° C. for 20 min.Alternatively, Zn dust (0.1 mmol) and ammonium formate (0.1 mmol) can beused. The reaction volume is reduced using a nitrogen stream, dilutedwith methylene chloride (0.5 mL), loaded onto a silica gel Sep-Pak, andeluted with 10% methanol in methylene chloride to give the final productfree of starting material.

Example 8 Synthesis of [F-18]-1-methyl-1-(4-fluorophenyl)piperaziniumsalt

t-BOC-Protected methylpiperizine was heated in the presence of1-fluoro-4-nitrobenzene under pressure in benzene to give4-t-BOC-protected 1-methyl-1-(4-nitrophenyl)piperazinium salt. Thepiperazinium salt was heated in the presence of potassium [¹⁸F]fluorideand Krytofix at 200° C. for 10 minutes. The oil was treated with aq. 3 MHCl for 20 minutes to give[F-18]-1-methyl-1-(4-fluorophenyl)piperazinium chloride.

Example 9 Synthesis of[F-18]-1-(4-fluorocyclohexyl)-1-methyl-4-phenylpiperazinium iodide

A solution of 1,4-cyclohexadiol (cis/trans mixture, 10 g, 86 mmol),benzoyl chloride (12 g, 86 mmol), and DMAP (50 mg) in methylenechloride/pyridine (80/20) was stirred at 25° C. for 4 hr. The reactionmixture washed with 10% HCl until the aqueous layer was acidic andmethylene chloride layer was washed with sat'd NaHCO₃, brine, and dried(Na₂SO₄). After solvent removal, chromatography (silica gel, methylenechloride/methanol, 95:5) of the crude material gave 6 g (30%) of thebenzoyl alcohol.

4-Benzoylcyclohexanol (5 g, 23 mmol) was oxidized using PCC (7.4 g,34.5) mmol) in methylene chloride (50 mL) for 2 hours at 25° C. Theblack reaction mixture was filtered through a short bed of silica geland solvent was removed. Chromatography (silica gel, hexane/ethylacetate, 85:15) afforded 3 g (60%) of the ketone; Mp 58-60° C.

Reductive amination to give 1-(4-benzoyoxycyclohexyl)-4-phenylpiperazinewas done using a published procedure [J. Org. Chem. 1996, 61,3849-3862]. 1-Phenylpiperazine (1 g, 6.2 mmol) and4-benzoyoxycyclohexanone (1.4 g, 6.2 mmol) were mixed in1,2-dichoroethane (35 mL) and then treated with sodiumtriacetoxyborohydride (1.2 g) and glacial acetic acid (0.25 g). Themixture was stirred at 25° C. under nitrogen for 16 hours. The reactionmixture was quenched by adding 1 N NaOH (20 mL) and the product wasextracted with methylene chloride. The extract was washed with brine,dried (MgSO₄), and solvent was evaporated to give the crude free base.Chromatography (silica gel, methylene chloride, 95:5) afforded 1.8 g(80%) of 1-(4-benzoyoxycyclohexyl)-4-phenylpiperazine.

Deprotection of 1-(4-benzoyoxycyclohexyl)-4-phenylpiperazine (1 g, 2.7mmol) was done in methanol (25 mL) using 1M lithium hydroxide (5 mL) at80° C. for 1 hr, which gave 0.6 g (85%) of1-(4-hydroxycyclohexyl)-4-phenylpiperazine.

1-(4-Hydroxycyclohexyl)-4-phenylpiperazine (0.5 g, 1.9 mmol) wasconverted to the mesylate using methane sulfonyl chloride (0.23 g, 2mmol) in 10 mL of methylene chloride/pyridine (90/10) at 25° C. Thereaction mixture was stirred for 2 hours and volatiles were removedunder vacuum. The crude product was dissolved in methylene chloride (20mL) and washed with sat'd NaHCO₃ twice and dried (MgSO₄). After solventremoval, the crude product was chromatographed on silica gel usingmethylene chloride/methanol (90:10), which gave 0.4 g (80%) of thepiperazine mesylate. ¹H NMR (CDCl₃), δ1.4-2.4 (9H, m, ring-CH), 2.7 (4H,m, piperazine-CH₂), 3.05 (3H, s, CH₃), 3.2 (4H, m, piperazine-CH₂), 4.6(1H, m, O—CH), 6.83-6.9 (2H, m, phenyl-CH), 7.2-7.3 (3H, m, phenyl-CH).

A Wheaton 5-mL reaction vial containing fluorine-18 (20 mCi) in 0.5 mLof ¹⁸O-enriched water, Kryptofix 2.2.2 (10 mg), and potassium carbonate(2 mg) was heated at 118° C. and solvent was evaporated with the aid ofa nitrogen gas flow. The K¹⁸F/Kryptofix complex was dried three times bythe addition of 1 mL of acetonitrile followed by evaporation of thesolvent using a nitrogen flow. A solution of 5 mg of1-(4-methanesulfonyoxycyclohexyl)-4-phenylpiperazine in 1 ml ofacetonitrile was added to the vial and the fluorination reaction wasperformed at 120° C. for 10 min. Solvent was removed using a nitrogenflow and replaced with 1 mL of a hexane/ethyl acetate/methanol (50:45:5)solution. After mixing, the solution was loaded onto a silica gel SepPak(Waters, Milford, Mass.) and the activity was eluted with 2 mL of thesame solution. The labeled piperazine derivative was purified by HPLC(semi-prep silica gel column, hexane/ethyl acetate/methanol, 50:45:5).The solvent was removed and iodomethane (0.1 mL) in acetonitrile (1 mL)was added to a vial containing the activity. The reaction vial was heatat 120° C. for 20 minutes and solvent was evaporated to afford[F-18]-1-(4-fluorophenyl)-1-methylpiperazinium iodide (7 mCi).

Example 10 Synthesis of[F-18]-1-(3-fluorocyclobutyl)-1-methyl-4-phenylpiperazinium iodide

Cyclobutanone-4-benzyloxy ether was prepared by a published procedure[J. Am. Chem. Soc. 1971, 93, 130; Bull. Chem. Soc. Jpn, 1988, 57, 1637].Reductive amination to give 1-(3-benzoxycyclobutyl)-4-phenylpiperazinewas done using the published procedure [J. Org. Chem. 1996, 61,3849-3862] use for the cyclohexyl analog. Hydrogenation, formation ofthe mesylate, and radio-labeling was done as described above.

Example 11 Synthesis of [F-18]-(4-fluorophenyl)triphenylphosphoniumnitrate (FTTP)

Non-radioactive standards and compounds used for radiolabeling wereprepared by the method of Homer [Chem. Ber. 1958, 91, 45] and Rieke [J.Am. Chem. Soc. 1976, 98, 6872]. 4-Nitroaniline (2.8 g, 0.02 mol) and themolar equivalent of sodium nitrite were dissolved in 10 ml ofconcentrated HCl acid 10 ml of water at 0° C. Water (20 ml), in whichwas dissolved sodium acetate (5.6 g), was added. Triphenylphosphine (5.6g) dissolved in ethyl acetate (80 ml) was added dropwise with stirring.After one hour, the resulting solution was acidified, the water layerseparated from the ethyl acetate, and the aqueous portion extractedtwice with ether. The ethyl acetate solution was extracted twice withwater, the extracts were combined with the other aqueous fraction.Addition of an aqueous solution of sodium iodide precipitated thephosphonium iodide, mp 225-227° C. (Lit. 228.5° C.; Chem. Ber. 1958, 91,45). 4-Nitrophenylphosphonium iodide was dissolved in 5 mL of ethanoltreated with an aqueous solution of AgNO₃. The silver iodide was removedby filtration and the solution was evaporated to dryness. Chromatographyof the crude salt on silica gel (methylene chloride/methanol (90:10))afforded pure 4-nitrophenylphosphonium nitrate; mp 215-127° C. Anal.calcd for C₂₄H₁₉N₂O₅P: C, 64.57; H, 4.29; Found: C, 64.49; H, 4.14.

[¹⁸F]FTPP was prepared from 4-nitrophenyltriphenylphosphonium nitrateand [¹⁸F]fluoride by nucleophilic aromatic substitution. A Wheaton 5-mLreaction vial containing fluorine-18 (600 mCi) in 1 mL of ¹⁸O-enrichedwater and 100 μL of ammonia hydroxide was heated at 120° C. and waterwas evaporated to near dryness (about 25 μL) with the aid of a nitrogengas stream. A 1 mL solution of the nitro compound in acetonitrile wasadded to the vial and water and solvent were removed by evaporation. Thecontents were dried three times by the addition of 1 mL of acetonitrilefollowed by evaporation of solvent using a nitrogen flow. The reactionvial was heated at 200° C. for 10 min, cooled to 25° C., and thecontents were dissolved in 1 ml of a 50% 0.1M Ca(NO₃)₂ in acetonitrile.The solution was loaded onto a alumunia SepPak (Waters, Milford, Mass.)to remove unreacted fluoride and the product purified by HPLC(Waters:Bondapak C-18, 19×150 mm column, flow: 6 mL/min; eluent: 50:50acetonitrile/aqueous 0.01M H₃PO₄). Solvent was removed byroto-evaporation and [¹⁸F]FTPP was dissolved in saline, the pH adjustedto 7.0 using sodium bicarbonate, and filtered (0.22 μm,Millipore:Millex-GV). Synthesis was completed within two hours; yield of[¹⁸F]FTPP was 20 mCi (6% EOB).

Example 12 Synthesis of[F-18]-(4-fluoro-3-nitrophenyl)triphenylphosphonium nitrate

The meta nitro analog was prepared in the same manner as that describedfor [F-18]-(4-fluorophenyl)triphenyl-phosphonium nitrate.

[¹⁸F]FTPP was prepared from (4-nitrophenyl)triphenylphosphonium nitrateand [¹⁸F]fluoride by nucleophilic aromatic substitution. A Wheaton 5-mLreaction vial containing fluorine-18 (600 mCi) in 0.5 mL of ¹⁸O-enrichedwater and 100 μL of ammonia hydroxide was heated at 120° C. and waterwas evaporated to near dryness (˜25 μL) with the aid of a nitrogen gasstream. A 1 mL solution of the nitro compound in acetonitrile was addedto the vial and water and solvent were removed by evaporation. Thecontents were dried three times by the addition of 1 mL of acetonitrilefollowed by evaporation of solvent using a nitrogen flow. The reactionvial was heated at 200° C. for 10 min, cooled to 25° C., and thecontents were dissolved in 0.5 ml of acetonitrile. The solution wasloaded onto a silica SepPak (Waters, Milford, Mass.) to remove unreactedfluoride and the crude product was eluted with 10% methanol in methylenechloride (4 mL). After removal of the solvent by evaporation, theresidue was dissolved in 50/50 acetonitrile:aqueous 0.01M H₃PO₄ andpurified by HPLC (Waters:Bondapak C-18 19×150 mm column, flow: 6 mL/min;eluent: same solvent). Solvent was removed by roto-evaporation and[¹⁸F]FTPP was dissolved in saline, the pH adjusted to 7.5 using sodiumbicarbonate, and filtered (0.22 μm, Millipore:Millex-GV). Synthesis wascompleted within two hrs; yield of [¹⁸F]FTPP was 10 mCi (3% EOB).

Example 13 Synthesis of [I-125]-p-iodophenyltriphenylphosponium nitrate(ITPP)

Synthesis of I-125 labeled 4-iodophenyltriphenylphosphonium (ITPP)regiospecifically from the triazene,4-piperidinylazophenyltriphenylphosphonium iodide, was reported by Shoupand Elmaleh. Triazenes have been well utilized for the preparation ofhigh specific activity receptor probes and this route offers high yieldsand purity. Conversion of 4-iodoaniline to the triazene was done bytrapping the diazonium ion with piperazine. The triazene was treatedwith triphenylphosphine and a catalytic amount of palladium (II) acetateto give the final precursor for I-125 labeling.

A mixture of 5.5 g (25.1 mmol) of 4-iodoaniline in 48 mL 6N—HCl wascooled in the ice-salt bath. To the mixture was added a precooledsolution of 1.89 g (27.4 mmol) of NaNO₂ in 12 mL H₂O. After stirring for10 min., an ice cold solution of 5.81 mL (58.7 mmol) of piperidine in10.5 g of KOH in 90 mL H₂O was added and stirring was continued. After10 min., ammonia was added until it became basic and the product wasextracted into CH₂Cl₂:EtOAc (1:1). The crude compound was purified bysilica gel column chromatography by 30% methylene chloride in hexaneelution to give 2.1 g (25.4%). An analytical sample was recrystallizedfrom hexane: mp 63-65° C. NMR: d 1.70 (s, 6H), 3.78 (s, 4H), 7.19 and7.63 (d, J=8.7 Hz, 4H). Anal. Calcd for C₁₁H₁₄IN₃: C, 41.92; H, 4.48; N,13.33; I, 40.27. Found: C, 42.08; H, 4.60; N, 12.99; I, 4.075.

Palladium (II) acetate (4.48 mg, 0.02 mmol) was added to a solution of630 mg (2 mmol) of triazene and 524 mg (2 mmol) of triphenylphosphine inxylene and the mixture was stirred at 110° C. overnight. The precipitatewas filtered off and washed with benzene, and dried to give 167 mg(14.5%) as a solid which was used without further purification. Ananalytical sample was prepared by further purification on silica gelcolumn by eluting with methylene chloride and recrystallization fromCH₂Cl₂-Ether: mp 255-257° C. NMR: d 1.75 (m, 6H), 3.96 (m, 4H), 7.72 (m,19H). Anal. calcd for C₂₉H₂₉IN₃P: C, 60.32; H, 5.06; N, 7.28; I, 21.98;P, 5.36. Found: C, 59.70; H, 5.14; N, 7.33; I, 21.65; P, 5.30.

Sodium ¹²⁵I (340 μCi) was dried in a Reaction vial under azeotropicconditions with MeCN at 110° C. under a stream of nitrogen. 600 μg oftriazene from the previous reaction, 30 μL of MeCN, and 5 μL of ClSiMe₃were added to the vial and the mixture was heated at 60° C. After 15min. 100 μL of NaHCO₃ solution was added and the reaction mixture wasextracted with CH₂C₂. The organic phase was washed three timesthoroughly with 20% AgNO₃ solution. After evaporating the solvent, thecrude product was dissolved in 0.1 mL CH₂Cl₂ and passed through a silicagel plug. This mini column was eluted with CH₂Cl₂ and acetone,respectively. The acetone fraction gave the desired product which washomogeneous on Radio-TLC (10% MeOH in CH₂Cl₂). The radiochemical yieldwas 67% and the specific activity was 17.4 Ci/mg.

Example 14 Cell and Biodistribution of[I-125]-p-iodophenyltriphenylphosponium nitrate (TPPI)

The biodistribution of TPPI in rats (6 per time point) at 5, 30, and 60minutes is presented in FIG. 2. Heart activity was over 0.7% dose pergram. The activity remained constant in the heart for a period of 60 minas expected from a microsphere. The activity washed fast from blood andheart-to-blood ratio increased from 2.9, 8.3 to 12.1 respectively.Heart-to-lung ratio changed from 0.96, 1.62 to 1.5. The heart density istwice that of the lung allowing for clear imaging of the heartespecially in tomographic imaging.

The cell distribution of TPPI mimics that of its tritiated analog; thecompound was accumulated and retained by carcinoma cells. High retentionwas observed in MCF-7 and C-6BAG glioma for a period of 240 minutes. TheC6 glioma cell line showed partial washout from the maximum accumulationat 30 minutes. The CV-1 normal cell line stayed constant with nosignificant uptake. FIG. 3 indicates that both tritiatedtetraphenylphosphonium and its radioiodinated version TPPI behave in thesame manner.

The biodistribution of TPPI in rats implanted with C6-BAG glioma showsthat the radioiodinated accumulated in the tumor tissue. Thetumor-to-brain ratio at 60, 120, and 240 minutes was 40, 17 and 44,respectively. Tumor-to-blood ratios were 12 at all times. Thelower-brain-to-tumor ratio measured at 120 minutes was due to the smallaverage tumor size of this group. In general, brain uptake could beassociated in part with breakdown of the blood-brain barrier. Sincethere was no washout from the tumor at 240 minutes, it is reasonable toassume that the high observed retention is at least in part due to theselective uptake mechanism of TPPI. FIG. 4 summarizes thebiodistribution of TPPI in rats implanted with C6-BAG glioma in brain.Uptake is expressed in % dose/gram.

Example 15 Cell and Biodistribution of[F-18]-(4-fluorophenyl)triphenylphosphonium nitrate (FTTP)

FTTP in saline (50-75 μCi) was injected directly into the femoral veinof each non-anesthetized rat and the animals were sacrificed andevaluated at five time points: 5, 30, and 60 min (six rats per timepoint). Blood was obtained by cardiac puncture. Syringes will be weighedbefore and after injection to determine the volume delivered. Theactivity per unit volume was obtained from standards. A total of eightdifferent tissues (blood, bone, lung, liver, kidneys, heart, muscle, andwhole brain) were excised, weighed and counted with a Packard Cobra IIAuto-Gamma Counter (Packard Instrument Co., Downers Grove, Ill.). Theraw counts were decay-corrected. All results are expressed as thepercentage injected dose per gram (% ID/g; mean±SD)

FIG. 5 shows the biodistribution of FTPP at 5, 30 and 60 min afterintravenous administration in rats (5 per time point). At 5 min,accumulation of FTPP in the heart (1.64 ID/g) was 11-fold higher than inblood and 5-fold higher than in liver. Accumulation of FTPP in lungs,liver, and kidneys was greater than in blood, muscle, and brain. At 30min, uptake of radioactivity in the heart was 1.5 ID/g and theheart-to-blood ratio was 75:1 (Table 1). Blood activity changedsignificantly from 5 to 60 min. Lung activity was 0.69 ID/g at 5 min,0.30 ID/g at 30 min, 0.38 ID/g at 60 min, while liver uptake was 0.18ID/g at 5 min, 0.17 ID/g at 30 min, 0.17 ID/g at 60 min. Heart-to-lungratios at 5, 30 and 60 min were 2.4, 5.0, 3.2 and, respectively. Boneaccumulation, an indication of defluorination, was minimal; 0.31 ID/g at5 min and increased to 0.39 ID/g after 60 min.

TABLE 1 Heart-to-tissue ratios for FTTP at 5, 30, and 60 min postadministration Ratios (% ID/g) 5 min 30 min 60 min Heart/Blood 11 75 70Heart/Lung 2 5 4 Heart/Liver 5 8 8

Example 16 PET Imaging with [F-18]-(4-fluorophenyl)triphenylphosphoniumnitrate (FTTP)

Rats (250-300 grams) or rabbits (3-4 Kg) were anesthetized and placedventrally to the imaging position and 0.4-1 mCi of the FTPP was injectedinto the vain. Three-dimensional dynamic data were acquired in list modefor one hour starting from the injection of the radiolabeled ligand.

PET imaging was conducted with a microPET, P4 system (ConcordeMicrosystems Inc, Knoxville, Tenn.). The length of the field of view is8 cm and the diameter is 22 cm allowing entire upper body imaging of therat or rabbit during a single acquisition. The imaging parameters ofthis system are in-plane and axial resolution of 1.2 mm full width ofphotopeak measured at half maximal count. MicroPET imaging of bothanimal species showed heart uptake after injection of FTPP with aninitial spike of activity corresponding to blood flow followed by aplateau after 1 min. FIG. 2 is a representative images of midlevel axial(left), coronal (middle), sagittal (left) views collected at 30-31 minpost FTPP administration and typical blood and tissue time-activitycurves obtained from sequential imaging for a period of one hour in arat.

FIGS. 10 and 11 are several heart tomographs obtained from a rabbitbefore and after LAD occlusion performed on the next day in the sameanimal. The rabbit was anesthetized, placed in the microPET camera (bodymarks in the camera positioning were made) and sequential images wereobtained for ten minutes following the administration of 3 mCi ofN-13-ammonia (FIG. 10). One hour later, FTPP was injected followed by 60minute sequential imaging of the rabbit (FIG. 11). Image corrections forthe remaining N-13-ammonia activity were made with an appropriateprogram. One day later, the same rabbit underwent an LAD occlusionprotocol, positioned in the same camera field of view and the above dualagent imaging sequence was repeated. FIGS. 10 and 11 represent severalheart levels and time-activity curves of the normal rabbit on the firstday injected with N-13-ammonia and FTTP, respectively. FIGS. 12 and 13are the imaging results obtained after LAD occlusion in one sectionaffected by occlusion. The time-activity curves and images clearlyindicate the area of diminished activity in the LAD occlusion. The highquality rabbit images show similar clearer delineation of the heartmuscle with FTTP compared to N-13-ammonia, an established myocardialperfusion agent. Late images at 60 min exhibited high myocardialretention of FTTP.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of obtaining a positron emission image of aportion of a mammal, measuring blood flow in the heart of a mammal, ormeasuring membrane transport in a mammal, comprising the steps of:administering to a mammal a compound represented by formula V; andacquiring a positron emission spectrum of a portion of said mammal,wherein the compound represented by formula V is:

wherein R¹ represents independently for each occurrence optionallysubstituted phenyl; R² is halogen-substituted phenyl, wherein saidhalogen substituent is ¹⁸F; and X is an anion that has an overall chargeof −1.
 2. The method of claim 1, wherein said compound has aradioactivity of greater than or equal to about 1 Curie/mmol.
 3. Themethod of claim 1, wherein said compound has a radioactivity of greaterthan or equal to about 100 Curie/mmol.
 4. The method of claim 1, whereinR¹ is phenyl.
 5. The method of claim 1, wherein R² is4-¹⁸F-fluorophenyl.
 6. The method of claim 1, wherein R¹ is phenyl andR² is 4-¹⁸F-fluorophenyl.
 7. The method of claim 1, wherein X is halide,acetate, or nitrate.
 8. The method of claim 1, wherein R¹ is phenyl, R²is 4-¹⁸F-fluorophenyl, and X is nitrate.
 9. The method of claim 1,wherein said mammal is a human, mouse, rat, dog, feline, monkey, guineapig, or rabbit.
 10. The method of claim 1, wherein said mammal is ahuman.
 11. A method of preparing (i) a fluorosubstituted aryl compound,or (ii) a fluorosubstituted alkenyl compound comprising the step of:reacting potassium fluoride with (i) a nitro aryl compound, wherein thenitro aryl compound does not contain an activing group or anelectron-withdrawing group to enhance the reactivity of the nitro group,thereby forming a fluorosubstituted aryl compound that does not comprisea nitro group, or (ii) an alkenyl halide or an alkenyl acetate, therebyforming a fluorosubstituted alkenyl compound.
 12. The method of claim11, wherein the reaction is conducted in the presence of a crown ether.13. The method of claim 12, wherein the crown ether is selected from thegroup consisting of 1,4,10-trioxa-7,13-diaza-cyclopentadecane;4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane;4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tricosane; and4,7,13,18-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane.
 14. The method ofclaim 11, wherein the potassium fluoride is potassium fluoride-18. 15.The method of claim 11, wherein the reaction is conducted undermicrowave irradiation.
 16. The method of claim 15, wherein the reactionis run with or without a solvent.
 17. The method of claim 15, whereinthe reaction is run with or without a catalyst.
 18. The method of claim1, for obtaining a positron emission image of a portion of a mammal. 19.The method of claim 11, for preparing a fluorosubstituted alkenylcompound.